Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Under a previous R21 grant the idea was pursued that lab adaptation of parasite strains would select for an increase in strain-independent virulence factors, such as extracellular survival, increased motility and accelerated replication cycles. Over 750 in vitro generations, we observed fast lab adaptation, especially in coping with extracellular conditions. At discrete points along the evolutionary path we tracked genomic level mutations by whole genome sequencing and gene expression changes by RNA-Seq. To our surprise, instead of genomic mutations, adaptations occurred all at the transcriptional level. Gene Set Enrichment Analysis (GSEA) revealed that changes in gene expression tracked with previous observations on in vitro induced differentiation to the bradyzoite stage. This fits with the hypothesis that extracellular parasites represent a stress-induced state in between tachyzoites and bradyzoites. The pivotal mechanism underlying changes in Toxoplasma gene expression are epigenetic changes mediated by a 67-member family of limitedly understood ApiAP2 transcription factors. Under this proposal the experimental (Gubbels) and computational (Zarringhalam) PIs will capitalize on the already established in vitro evolved series of parasites to unravel the transcriptional programs underlying lab adaptation. Under Aim 1 we will establish chromatin accessibility by using Assay for Transposase-Accessible Chromatin sequencing (ATAC-Seq) as a proxy for transcription factor binding along the evolutionary path, which has been robustly demonstrated in the closely related Plasmodium parasites and is unbiased on the nature of both cis- and trans-elements. We will develop a novel computational pipeline to correlate changes in chromatin access with changes in gene expression along the evolutionary path. This will identify a set of cis-elements in promoters of gene clusters associated with lab adaptation and differentiation. In Aim 2 we will computationally narrow down the set of cis- elements to the most significant and experimentally validate the cis-acting transcriptional power through a Nano luciferase transcriptional reporter system. Thus, we will be able to connect changes in expression of gene clusters with the identity of cis-acting elements, whose nature has remained quite elusive (spare a handful of success stories). Notably, our approach bypasses a key roadblock in Toxoplasma ApiAP2 research: the short cell cycle (6 hrs) and poor cycle synchronization tools have prevented success in characterizing genes expression controls, which we overcome by studying highly synchronous extracellular parasites in a non- proliferative state. Upon successful completion of this proposal we will advance the molecular basis Toxoplasma virulence, and provide a map of cis-regulatory motifs on development of virulence traits, including programs shared with bradyzoite differentiation. These insights will provide starting points toward new avenues for developing anti-toxoplasmosis agents.